Composition and method for inhibition of microorganisms

ABSTRACT

A composition and method for the inhibition of microorganisms.

CROSS-REFERENCE TO RELATED APPLICATION

This application is a divisional application of U.S. patent applicationSer. No. 10/535,357, filed May 18, 2005, which is a U.S. nationalcounterpart application of international application serial No.PCT/US2003/037526 filed Nov. 24, 2003, which claims priority to U.S.Provisional Patent Application No. 60/428,863 filed Nov. 25, 2002. Theentirety of which is hereby incorporated by reference.

FIELD OF THE INVENTION

The present invention relates generally to a composition and method forthe inhibition of microorganisms, and more particularly to a compositionand method for controlling Listeria monocytogenes in food processingfacilities or on a food product by, for example, a probioticmicroorganism, such as competitive exclusion microorganisms.

BACKGROUND OF THE INVENTION

The control of contamination by microorganisms is a recognized problemin the food processing industry. The process of preparing food productsis largely concerned with preventing the contamination of such foodproducts with harmful microorganisms. For example, in the meat packingindustry, many types of microorganisms can cause food poisoning ifcontamination takes place. These microorganisms include E. coli,Salmonella, Listeria monocytogenes, Staphylococcus aureus, Bacillusanthracis, Campylobacter coli, Campylobacter jejuni, Yersiniaenterocolitica, Yersinia pseudo tuberculosis, Brucella, and Clostridium.One microorganism of particular concern for the food processing industryis Listeria monocytogenes (hereinafter referred to as L. monocytogenes)since studies indicate that certain strains of L. monocytogenes canbecome well established in a food-processing facility and remain membersof the resident microbial flora for months or years. Moreover,investigations of several outbreaks of listeriosis revealed thatenvironmental contamination of food processing facilities was theprimary source of L. monocytogenes in many commercially preparedready-to-eat (RTE) processed foods.

In general, L. monocytogenes has a widespread occurrence in nature andis capable of surviving and growing under a variety of conditions,including growing in soil and aqueous environments. For example, L.monocytogenes has been isolated from 8.4 to 44% of samples obtained fromgrain fields, pastures, mud, animal feces, wildlife feeding grounds, andrelated sources, and can survive in moist soils for more than 295 days.Furthermore, L. monocytogenes is a nonfastious organism that thrives incool, damp environments. Moreover, this organism can grow attemperatures typically used to refrigerate processed foods whichpresents particular problems for the food processing industry.

As previously mentioned, L. monocytogenes thrives in cool, dampenvironments which is why high populations of this organism frequentlyoccur in floor drains of food processing facilities. These L.monocytogenes-contaminated floor drains can serve as a point ofcontamination for the processing plant environment and food products.Decontaminating floor drains of listerae is especially challengingbecause the population of L. monocytogenes is typically enveloped in abiofilm, and the biofilm provides the microorganism with unusualprotection against disinfectants and conventional treatments availableto control pathogens on environmental surfaces. Therefore, althoughmajor improvements have been made in food processing plant layout,equipment design, and in procedures for cleaning and sanitizing foodprocessing facilities, controlling the widely distributed psychrotrophicL. monocytogenes in food processing facilities remains a formidablechallenge for the entire food industry as demonstrated by the fact thatenvironmental testing results indicate that L. monocytogenes continuesto be introduced into food processing environments. Accordingly, acomposition and method for the inhibition of microorganisms such as L.monocytogenes is desirable.

SUMMARY OF THE INVENTION

According to one illustrative embodiment, there is provided a method oftreating a surface of a food processing facility which has a firstpopulation of microorganisms disposed thereon. The method includesdisposing (i) a biofilm containing a second population of microorganismsand/or (ii) a second population of microorganisms that forms a biofilmonto the surface of the food processing facility. The method alsoincludes inhibiting the growth of the first population of microorganismson the surface of the food processing facility with the secondpopulation of microorganisms.

According to another illustrative embodiment, there is provided a methodof inhibiting the growth of Listeria monocytogenes on a surface of afood processing facility. The method includes inoculating the surface ofthe food processing facility with antimicrobial bacteria that can adhereto surfaces. The method also includes inhibiting the growth of theListeria monocytogenes on the surface of the food processing facilitywith the bacteria adhering to the surface. For example, the bacteria canbe contained in a biofilm so that it adheres to the surface or thebacteria can be one that forms a biofilm such that it adheres to thesurface.

According to another illustrative embodiment, there is provided a methodof inhibiting the growth of Listeria monocytogenes on a surface of afood processing facility. The method includes inoculating the surface ofthe food processing facility with a microorganism selected from thegroup consisting of bacteria from the genus Enterococcus and bacteriafrom the genus Lactococcus. The method also includes inhibiting thegrowth of the Listeria monocytogenes on the surface of the foodprocessing facility with the bacteria.

According to still another illustrative embodiment, there is provided amethod of inhibiting the growth of Listeria monocytogenes on a surfaceof a food processing facility. The method includes inoculating thesurface of the food processing facility with antimicrobial bacteria thatcan adhere to surfaces such as antimicrobial bacteria contained in abiofilm, wherein the bacteria are selected from the group consisting ofEnterococcus durans, Lactococcus lactis, and Lactobacillus plantarum.The method also includes inhibiting the growth of the Listeriamonocytogenes on the surface of the food processing facility with thebacteria contained in the biofilm.

According to yet another illustrative embodiment, there is provided akit for inhibiting the growth of a first microorganism populationdisposed on a surface. The kit can include a biofilm and a secondmicroorganism population disposed in the biofilm. In the alternative,the kit can include a microorganism capable of forming a biofilm whendisposed on a surface. In another alternative the kit can include abiofilm and a microorganism, where the microorganism is placed in thebiofilm prior to being disposed on the surface. The second microorganismpopulation is inhibitory to the first microorganism population when thesecond microorganism population is placed in the presence of the firstmicroorganism population.

According to still another illustrative embodiment, there is provided aninoculant composition. The inoculant composition includes a biofilmhaving disposed therein at least one of the following: Enterococcusdurans 141-1 having ATCC accession number PTA-4758, Enterococcus durans152 having ATCC accession number PTA-4759, Lactococcus lactis C-1-92having ATCC accession number PTA-4760, or Lactococcus lactis C-1-152having ATCC accession number PTA-4761 or mixtures thereof. Each of saidstrains having been deposited with the American Type Culture Collection(ATCC; 10801 University Blvd., Manassas, Va. 20110-2209) on Oct. 15,2002.

According to yet another illustrative embodiment, there is provided abiologically pure culture of bacteria selected from Enterococcus durans141-1 having ATCC accession number PTA-4758, Enterococcus durans 152having ATCC accession number PTA-4759, Lactococcus lactis C-1-92 havingATCC accession number PTA-4760, or Lactococcus lactis C-1-152 havingATCC accession number PTA-4761.

According to still another illustrative embodiment, there is provided akit for inhibiting the growth of a first microorganism populationdisposed on a surface. The kit includes a biofilm and a secondmicroorganism population for disposing in the biofilm. The secondmicroorganism population is inhibitory to the first microorganismpopulation when the second microorganism population is placed in thepresence of the first microorganism population.

According to yet another illustrative embodiment, there is provided amethod for selecting bacteria which inhibit the growth of Listeriamonocytogenes. The method includes isolating naturally-occurringbacteria from a food processing facility. The method also includesculturing the isolated naturally-occurring bacteria. The method furtherincludes testing the isolated naturally-occurring bacteria for theability to inhibit the growth of Listeria monocytogenes.

According to still another illustrative embodiment, there is provided amethod of selecting inhibitory bacteria. The method includes isolatingnaturally-occurring bacteria populations from a food processingfacility. The method also includes culturing the isolatednaturally-occurring bacteria populations. The method further includestesting each isolated naturally-occurring bacteria population for theability to inhibit the growth of a microorganism, where isolatednaturally-occurring bacteria populations having the ability to inhibitthe growth of the microorganism are identified as a population ofinhibitory bacteria.

According to still another illustrative embodiment, there is provided aculture of microorganisms that includes Enterococcus durans having ATCCaccession number PTA-4758.

According to still another illustrative embodiment, there is provided aculture of microorganisms that includes Enterococcus durans having ATCCaccession number PTA-4759.

According to still another illustrative embodiment, there is provided aculture of microorganisms that includes Lactococcus lactis having ATCCaccession number PTA-4760.

According to still another illustrative embodiment, there is provided aculture of microorganisms that includes Lactococcus lactis having ATCCaccession number PTA-4761.

According to still another illustrative embodiment, there is provided amethod of treating a food product having a first population ofmicroorganisms disposed thereon. The method includes (a) disposing asecond population of microorganisms onto the surface of the food productand (b) inhibiting the growth of the first population of microorganismson the food product with the second population of microorganisms. Ifdesired, the second population of microorganisms can be capable offorming a biofilm.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 illustrates the results of analyzing four probiotic isolates fortheir DNA fingerprinting by pulsed field-gel electrophoresis; lane 1Lambda ladder DNA standard, lane 2 C-1-92 (L. lactis subsp. lactis),lane 3 C-1-152 (L. lactis subsp. lactis), lane 4 141-1 (E. durans), andlane 5 152 (E. durans).

DETAILED DESCRIPTION

While the invention is susceptible to various modifications andalternative forms, specific exemplary embodiments thereof have beenshown by way of example in the drawings and will herein be described indetail. It should be understood, however, that there is no intent tolimit the invention to the particular forms disclosed, but on thecontrary, the intention is to cover all modifications, equivalents, andalternatives falling within in the spirit and scope of the invention.

Materials and Methods

Bacterial strains: A five-strain mixture of L. monocytogenes, includingLM101 (serotype 4, salami isolate), LM112 (serotype 4, salami isolate),LM113 (serotype 4, pepperoni isolate), H9666 (serotype 1/2c, humanisolate) and ATCC 5779 (serotype 1/2c, cheese isolate) from the UGACenter for Food Safety culture collection were used. Each strain wasindividually grown in tryptic soy broth with 0.6% yeast extract (TSBYE,Becton Dickinson, Sparks, Md.) at 37° C. for 16 h. The cultures weresedimented by centrifugation at 8,000×g for 20 min and resuspended in0.1% peptone. The optical density of each strain was adjusted in aspectrophotometer with 0.1% peptone to an OD reading of 0.5 (ca. 10⁸cfu/ml) at 630 nm. An equal volume of culture of each of the fivestrains was combined to obtain a 5-strain mixture of approximately equalcell numbers of each strain.

Isolation and Screening of Microorganisms for Metabolites Antagonisticto L. monocytogenes

Biofilm samples collected from floor drains at different food processingplants having a recent history of no detectable L. monocytogenes wereused to obtain isolates of bacteria and yeasts. Two methods, whichincluded a direct plating and an enrichment culture procedure, were usedto isolate these microorganisms. Tryptic soy broth (TSB, 10 ml) wasadded to each biofilm sample (ca. 1 g) and biofilm preparations wereserially diluted (1:10) in 0.1% peptone to 10⁻³. A volume of 0.1 ml ofeach dilution was plated on dichloran rose bengal chloramphenicol agar(DRBC) and tryptic soy agar (TSA) plates in duplicate, with DRBC platesincubated at 30° C. for 72 h and TSA plates incubated at 37° C. for 24h. Biofilm preparation (1 ml) also was added to 9 ml of TSB andincubated at 37° C. for 24 h. Enrichment cultures were serially dilutedin 0.1% peptone and 0.1-ml portions of dilutions of 10⁻⁵ to 10⁻⁸ wereplated onto TSA and DRBC plates, and incubated according to theconditions described above. Ten colonies per biofilm specimen wereselected randomly from plates and streaked for isolation.

Two methods, including the spot-on-lawn assay and the double layerassay, and two temperatures (37° C. and 15° C.) were used to screenisolates for anti-listerial activity. For the spot on-lawn-assay, 0.1 mlof ca. 10⁷ cells of the 5-strain mixture of L. monocytogenes/ml wasplated onto each of duplicate TSA plates. Candidate competitiveexclusion inhibition isolates were grown individually in TSB at 37° C.for 24 h, cells were sedimented by centrifugation (4,000×g for 20 min),and the supernatant fluid of each culture was filter-sterilized(0.22-μm-pore-size cellulose acetate membrane; Nalgene Co., Rochester,N.Y.). A 12-mm disc (Dispens-O-Disc; Difco Laboratories, Detroit, Mich.)was placed onto the surface of each TSA plate, and 0.1 ml offilter-sterilized supernatant fluid from a single culture was applied tothe surface of the disc. The plates were incubated at 37° C. for 24 hand observed for zones of inhibition. In addition, a disc with nisin(3.125 μg; Sigma, St. Louis, Mo.) was used as the positive control and adisc with 0.1 M phosphate-buffered saline (PBS), pH 7.2, was used as thenegative control.

The double-layer assay, also a two-step procedure, involved firstgrowing a spot-inoculated candidate competitive inhibition isolate onTSA and then applying a second layer of growth medium containing the5-strain mixture of L. monocytogenes. Specifically, an individual colonyof competitive bacteria was inoculated in the center of each of two theTSA plates and incubated for 24 h at 37° C. A 5-strain mixture of L.monocytogenes was added at 10⁶ cfu/ml to brain heart infusion with 0.4%agar (BHIA, Difco) at 50° C. and mixed for 1 min at 200 rpm with amagnetic stir bar. The mixture (8 ml) was poured onto each TSA plate asa second layer and allowed to cool to room temperature. The cultureswere incubated for 24 h at 37° C. and observed for zones of inhibition.Nisin-producing Lactococcus lactis subsp. lactis (ATCC 11454) was usedas the positive control and a yeast isolate, which was obtained fromthis study and confirmed to have no inhibitory effect on the growth ofL. monocytogenes was used as the negative control.

Competitive Inhibition in Broth Cultures at Different Temperatures

All isolates having anti-L. monocytogenes activity were further testedin TSBYE for competitive growth at temperatures ranging from about 4° C.to about 37° C., for example, 4° C., 8° C., 15° C. or 37° C. Anindividual isolate of a candidate competitive microorganism at ca. 10⁷to 10⁹ (range 10^(6.7)-10^(9.0)) cfu in 0.1 ml and 0.1 ml of ca. 10⁵ to10⁶ (range 10^(5.2)-10^(6.6)) cfu of a 5-strain mixture of L.monocytogenes were added to 10 ml of TSBYE and incubated at 4°, 8°, 15°or 37° C. Cultures (1 ml) were sampled at intervals of 0, 8 and 24 h forincubation at 37° C., at 0, 1, 2, 3, 7, 10 and 14 days for 15° C., at 0,1, 7, 14 and 21 days for 8° C., and at 0, 2, 7, 14, 21 and 28 days for4° C., and enumerated for L. monocytogenes on modified Oxford agar (MOX,Difco) at 37° C. for 48 h and for competitive microorganism count on TSAat 37° C. for 48 h.

Identification of Competitive Microorganisms

Bacterial isolates having antagonistic activity to L. monocytogenes atall four temperatures evaluated were assayed by Gram stain, biochemicalassays (API CHB and API CHL; bioMérieux Industry, I'Etoile, France), and16S rRNA gene alignment profile analysis (Midi Labs, Newark, Del.) foridentification of genus and species.

Preparation of stainless steel coupons: Stainless steel (T-304, TullMetals Company, Atlanta, Ga.) coupons (4 cm×2.5 cm) were washed by a10-min immersion with agitation (150 rpm) in 1000 ml of an aqueous 2%RBS 35 Detergent solution (20 ml of RBS 35 Concentrate per liter of tapwater at 50° C.; Pierce, Rockford, Ill.), and rinsed by immersion in1000 ml of tap water (initially at 50° C.) with agitation (150 rpm) for25 min. Five additional 1-min immersions with agitation (150 rpm) in1000 ml of distilled water at ambient temperature were performed. Thecoupons were dried and a hydrophobic marker was used to encircle an areaof 1.13 cm in diameter. The coupons were then individually wrapped inaluminum foil and autoclaved at 121° C. for 30 min.

Competitive inhibition of L. monocytogenes in a biofilm: Biofilms weregrown using a modification of the protocol described by Leriche andCarpentier, Viable but non-culturable Salmonella typhimurium in single-and binary-species biofilms in response to chlorine treatment. J. FoodProt. 58:1186-1191 (1995) which is incorporated herein by reference andChae and Schraft, Cell viability of Listeria monocytogenes biofilms.Food Microbiol. 18:103-112 (2001) which is incorporated herein byreference. The protocol was modified to include increasing the circle toa diameter of 1.13 cm and adjusting the incubation temperatures andsampling times to produce examples of the biofilms described herein. Abiofilm is defined as an assemblage, or organized group, of microbialcells, wherein the assemblage is irreversibly associated with a surfaceand enclosed in a matrix of primarily polysaccharide material. Aninoculum of 0.1 ml of 10^(6.0)-10^(8.4) cfu of candidate competitivemicroorganisms and 0.1 ml of 10^(2.6)-10^(4.6) cfu of a 5-strain mixtureof L. monocytogenes were deposited in a biofilm and placed within themarked area of the stainless steel coupon. The coupon was then placed ina humidity-controlled incubator (ca. 95% relative humidity) at 4°, 8°,15° or 37° C. for 6 h. Non-adherent bacteria were removed by vacuumaspiration after 6 h of incubation and replaced with 0.1 ml of freshTSB. The stainless steel coupons were reincubated at the sametemperature and the media were replaced every 7, 3, 3 and 1 days forincubation at 4°, 8°, 15° or 37° C., respectively. At each samplingtime, selected coupons in duplicate were transferred to a laminar flowhood in which weakly adherent bacteria were removed by washing themarked area of each coupon 3 times with PBS, then removing the remainingliquid from the marked area by vacuum aspiration. Each coupon was placedin a 50-ml centrifuge tube containing 9.9 ml of PBS and 30 glass beads(5 mm, Fisher Scientific, Norcross, Ga.) and agitated by a Vortex mixer(Fisher Scientific) for 2 min to disrupt bacteria in the adherentbiofilm. The suspended bacteria were serially diluted (1:10) in 0.1%peptone and plated in duplicate on TSA for enumeration of competitivemicroorganisms or total bacteria (if L. monocytogenes counts on MOX weregreater than or equal to the bacterial counts on TSA) and MOX for L.monocytogenes. The plates were incubated for 48 h at about 37° C. andcompetitive microorganism and L. monocytogenes counts were determined.Coupons inoculated with only 10^(2.6)-10^(4.6) L. monocytogenes servedas positive controls, whereas coupons inoculated only with10^(6.0)-10^(8.4) competitive microorganisms served as negativecontrols. Results reported were the average of duplicate determinations.

Identification of nisA and nisB competitive microorganisms: A polymerasechain reaction (PCR) method was used to identify competitivemicroorganisms that encode NisA and NisB. Bacterial DNA was extractedusing a microbial genomic DNA isolation kit according to the protocoldescribed by the manufacturer (Mo Bio Laboratories, Solana Beach,Calif.). The oligonucleotide sequences of the primers used for nisA were5-CGGCTCTGATTAAATTCTGAAG (SEQ ID NO:1) and 5-CGGTTGAGCTTAAATGAAC (SEQ IDNO:2) and for nisB were 5-AGAGAAGTTATTTACGATCAAC (SEQ ID NO:3) and5-ATCTGACAACAAATCTTTTTGT (SEQ ID NO:4). PCR was performed with anIcycler 96 Well Reaction Module (Bio-Rad Laboratories, Hercules, Calif.)according to the procedure described by Olasupo, N. A., U. Schillinger,A. Narbag, H. Dodd, and W. H. Holzapfel, Occurrence of nisin Zproduction in Lactococcus lactis BFE 1500 isolated from wara, atraditional Nigerian cheese product. Int. J. Food Mirobiol. 53:141-152(1999) incorporated herein by reference.

Results

A total of 12 biofilms from floor drains of four different foodprocessing facilities were screened for microorganisms inhibitory to L.monocytogenes. A total of 156 yeast and 257 bacterial isolates wereobtained from the biofilms and assayed for antagonistic activity againstL. monocytogenes. Twenty-four isolates, including 3 yeasts and 21bacteria, were inhibitory to L. monocytogenes (0.5 to 3.5 mm zones ofinhibition), with no bacteria and 3 yeasts identified by thespot-on-lawn assay and 21 bacteria and no yeast identified by thedouble-layer assay.

All isolates antagonistic to L. monocytogenes were evaluatedindividually for their ability to inhibit growth or inactivate a5-strain mixture of L. monocytogenes in TSB at about 37° C. Under theseconditions, two yeast isolates were weakly antagonistic to L.monocytogenes, repressing growth of listeriae by 0.7 log₁₀ cfu/mlcompared to the positive control of L. monocytogenes only. In contrast,nine of the biologically pure bacterial isolates were stronglyantagonistic to L. monocytogenes, with each providing a greater than 5log₁₀ cfu/ml differential at 24 h when compared with the L.monocytogenes—only positive control as shown in Table 1 below. Four ofthese bacterial isolates are deposited with the American Type CultureCollection, located at 10801 University Blvd, Manassas, Va. 20110-2209,on Oct. 15, 2002. In particular, the following microorganisms aredeposited with the ATCC, Enterococcus durans 141-1 having ATCC accessionnumber PTA-4758, Enterococcus durans 152 having ATCC accession numberPTA-4759, Lactococcus lactis C-1-92 having ATCC accession numberPTA-4760, or Lactococcus lactis C-1-152 having ATCC accession numberPTA-4761.

TABLE 1 Inhibition at about 37° C. of L. monocytogenes (LM) bycompetitive microorganism (CM) in tryptic soy broth L. monocytogenes orcompetitive microorganism count (log₁₀ cfu/ml) 0 time 8 h 24 h IsolateLM LM + CM LM LM + CM LM LM + CM No. only^(a) CM^(b) only^(c) only CMonly only CM only Bacteria C-2-101 3.0 3.0 5.7 6.7 6.4 7.4 9.4 9.0 8.4C-1-152 3.0 2.8 6.3 6.4 5.3 9.1 9.8 7.8 9.2 C-2-188 3.5 2.7 6.5 6.4 6.76.5 9.1 9.3 7.7 C-1-92 3.3 3.2 6.0 6.3 <0.7^(d) 9.5 9.3 0.7 9.3 143 3.22.9 6.1 6.5 1.7 9.1 8.8 <0.7 9.3 375-1 3.0 2.9 5.8 6.7 2.2 9.4 9.5 2.89.4 129 2.8 3.0 6.4 6.6 5.2 9.2 9.4 8.5 9.4 123 3.5 3.3 6.3 6.7 6.5 8.29.2 9.0 9.6 B-2-18 3.3 3.2 5.7 6.7 5.8 9.1 9.4 7.6 9.6 152 3.6 3.4 6.26.7 2.4 9.1 8.7 3.3 9.5 141 3.5 3.4 6.0 7.9 3.5 8.8 9.5 3.1 9.1 123 3.23.2 6.0 6.4 6.6 8.5 9.0 9.2 9.7 147 3.2 3.2 6.1 7.6 2.7 9.4 9.4 3.6 9.3375-3 2.9 2.8 5.8 5.7 5.9 7.7 9.3 6.9 9.4 107 3.0 3.1 6.1 6.7 6.1 8.69.6 6.7 9.0 (tiny) 123 3.2 3.2 6.3 6.7 6.2 8.2 9.6 8.3 9.6 375-2 3.3 3.16.3 6.4 2.0 9.4 9.4 3.1 9.3 107 3.3 3.2 6.0 7.2 6.4 7.8 9.4 7.7 9.2 141(2) 3.1 3.0 6.3 7.4 3.1 9.1 9.6 3.8 9.6 143 (2) 3.2 3.2 7.0 7.5 3.4 9.39.3 4.0 9.3 107 (2) 3.2 3.2 6.0 7.3 6.8 8.2 9.0 6.7 9.4 Yeast C-2-45 2.62.6 4.5 6.7 5.5 6.6 9.6 9.6 7.6 C-2-187 2.7 2.7 4.4 5.7 5.3 6.9 9.9 9.27.8 C-3-53 2.9 2.7 5.6 7.0 5.6 7.2 10.0 9.3 7.8 ^(a)LM only = L.monocytogenes count ^(b)LM + CM = L. monocytogenes count ^(c)CM only =Competitive microorganism count ^(d)Minimum detection limit 0.7 log₁₀cfu/ml

Twelve isolates were assayed under the same conditions, but at about 15°C. Three of the isolates were highly antagonistic to L. monocytogenes,with greater than a 4 log₁₀ L. monocytogenes differential at day 7compared to L. monocytogenes—only positive control, and one isolate,C-1-92, was exceptionally bactericidal, with no detectable L.monocytogenes present (>8 log₁₀ L. monocytogenes/ml differentialcompared with positive control) at 7 and 14 days, see Table 2 below.

TABLE 2 Inhibition at about 15° C. of L. monocytogenes (LM) bycompetitive microorganisms (CM) in tryptic soy broth L. monocytogenes orcompetitive microorganism count (log₁₀ cfu/ml) at day 0 time 1 d 2 d 7 d14 d Isolate LM LM + CM LM LM + CM LM LM + CM LM LM + CM LM LM + CM NoOnly^(a) CM^(b) only^(c) only CM only only CM only only CM only only CMonly 141 3.4 3.4 6.2 6.8 5.4 8.8 9.8 2.9 9.6 9.7 8.4 9.6 9.0 9.1 8.4141-2 3.4 3.4 6.2 7.3 2.7 9.6 9.7 2.8 9.6 9.8 7.2 9.8 9.0 7.6 9.8 B-2-183.5 3.3 6.7 7.2 6.3 9.6 9.8 7.2 9.9 9.4 8.0 10.2 9.6 8.0 10.2 152 3.43.4 6.2 7.2 6.0 9.1 9.8 4.1 9.4 9.7 7.3 9.4 9.1 8.6 9.4 143-2 3.5 3.45.5 7.4 7.2 8.8 9.8 4.7 9.5 9.6 8.5 9.7 8.8 8.1 9.6 147 3.5 3.4 6.3 7.52.6 9.4 9.9 2.4 9.6 9.7 7.4 9.7 9.3 8.5 9.7 375-2 3.6 3.3 6.4 7.8 2.39.5 9.9 3.0 9.8 9.9 6.6 9.5 8.7 7.7 9.9 129 3.5 3.2 5.5 6.6 5.5 9.8 10.35.1 9.8 9.6 6.9 9.3 9.0 8.6 6.9 C-1-92 3.4 3.3 6.4 8.0 1.7 9.9 10.2 1.09.8 9.2 <0.7^(d) 9.2 9.0 <0.7 7.8 375-1 3.4 3.4 6.3 6.5 6.2 9.1 9.7 3.09.7 9.3 3.8 9.4 9.1 7.2 9.8 C-1-152 3.5 3.4 6.3 7.9 7.1 9.4 9.9 8.0 9.59.7 8.2 9.3 9.0 9.5 9.4 143 3.6 3.4 6.4 7.2 7.0 8.0 9.7 3.5 9.5 8.3 4.19.3 8.5 7.6 9.5 ^(a)LM only = L. monocytogenes count ^(b)LM + CM = L.monocytogenes count ^(c)CM only = Competitive microorganism count^(d)Minimum detection limit 0.7 log₁₀ cfu/ml

These same twelve isolates were assayed for antagonistic activity to L.monocytogenes in TSB at about 8° C. Six of isolates were highlyinhibitory, with greater than a 4 log₁₀ L. monocytogenes/ml differentialat 14 days compared to the L. monocytogenes only positive control, andone isolate, 152, was exceptionally antimicrobial, with a 6.3 log₁₀ L.monocytogenes/ml differential at 21 days, see Table 3 below.

TABLE 3 Inhibition at about 80° C. of L. monocytogenes (LM) bycompetitive microorganisms (CM) in tryptic soy broth L. monocytogenes orcompetitive microorganism count (log₁₀ cfu/ml) 0 time 1 d 7 d 14 d 21 dIsolate LM LM + CM LM LM + CM LM LM + CM LM LM + CM LM LM + No. only^(a)CM^(b) only^(c) only CM only only CM only only CM only only CM onlyBacteria 141 3.3 3.3 6.1 4.0 4.0 6.4 8.9 4.4 9.3 9.8 5.2 9.5 9.8 6.4 9.4141-2 3.4 3.3 6.3 3.7 3.8 6.3 8.5 5.0 9.2 9.7 5.3 9.4 9.8 7.6 9.5 B-2-183.3 3.5 6.7 4.2 4.2 7.0 8.9 7.4 9.1 9.8 9.0 9.0 9.3 8.7 9.4 152 3.3 3.26.2 4.2 4.0 6.6 9.0 2.7 9.1 9.9 2.9 9.3 9.8 3.5 9.4 143-2 3.2 3.1 6.43.7 3.5 6.4 8.0 6.0 8.3 9.4 6.4 8.9 9.5 8.0 9.1 147 3.5 3.5 6.4 3.7 3.86.2 8.5 7.3 9.2 9.4 9.2 9.4 9.5 8.9 9.5 375-2 3.3 3.2 6.3 4.2 3.5 6.49.3 3.2 9.1 9.8 5.5 9.2 9.8 7.5 9.5 129 3.3 3.1 5.2 3.5 3.8 6.3 9.3 6.98.9 9.8 6.9 9.2 9.8 7.5 9.2 C-1-92 3.5 3.4 6.3 3.7 3.5 6.0 9.1 5.7 8.39.8 5.3 8.8 9.9 5.1 8.8 375-1 3.5 3.5 6.3 3.9 3.9 6.4 9.5 4.6 9.1 9.74.7 9.1 9.8 6.5 9.3 C-1-152 3.2 3.3 6.4 4.0 4.0 6.4 8.9 5.6 9.4 9.7 6.79.3 9.8 7.9 9.4 143 3.2 2.6 6.1 3.7 3.5 7.0 9.5 5.2 8.4 9.8 6.9 9.2 9.77.9 9.4 Yeast C-2-45 2.3 2.5 5.7 3.2 3.1 5.4 8.1 8.1 7.7 9.6 9.1 7.8 9.89.4 7.9 C-3-53 2.5 2.7 5.8 3.4 3.2 5.6 8.1 8.2 8.0 9.9 7.8 6.3 9.8 9.28.1 C-2-187 2.2 2.3 4.7 2.7 2.8 4.4 8.5 7.3 5.1 9.7 9.3 7.7 9.8 9.7 6.5^(a)LM only = L. monocytogenes count ^(b)LM + CM = L. monocytogenescount ^(c)CM only = Competitive microorganism count

Nine isolates with antagonistic activity to L. monocytogenes at allthree temperatures were assayed for their activity against L.monocytogenes at about 4° C. Three isolates were highly antagonistic,with greater than a 4 log₁₀ L. monocytogenes/ml differential at 28 dayscompared to the positive control, and one isolate, 152, wasexceptionally antimicrobial, with a 6 log₁₀ differential at 28 days, seeTable 4 below.

TABLE 4 Inhibition at about 4° C. of L. monocytogenes (LM) bycompetitive microorganisms (CM) in tryptic soy broth L. monocytogenes orcompetitive microorganism count (log₁₀ cfu/ml) 0 time 7 d 14 d 21 d 28 dIsolate LM LM + CM LM LM + CM LM LM + CM LM LM + CM LM LM + CM No.only^(a) CM^(b) only^(c) only CM only only CM only only CM only only CMonly 141-1 2.7 2.6 5.0 4.2 4.1 5.6 6.0 6.5 6.5 8.6 6.6 8.2 9.5 6.3 8.7152 3.4 3.4 6.4 4.1 3.7 6.6 7.0 4.3 7.5 8.5 4.5 8.7 9.9 3.9 9.2 C-1-923.5 3.0 6.5 4.1 3.7 5.9 6.9 5.8 5.7 8.2 6.7 5.0 9.9 8.0 5.3 143-1 2.92.5 5.0 3.3 3.3 5.2 6.7 5.9 5.2 8.0 7.5 6.4 9.5 8.3 6.9 C-1-152 2.7 2.95.9 3.7 3.5 5.6 6.5 6.1 6.6 8.3 7.1 7.3 9.5 7.4 8.4 375-1 2.8 2.4 5.24.2 3.8 5.4 7.0 6.4 6.7 8.4 7.2 8.0 9.7 7.3 8.6 143-2 3.3 3.2 5.9 4.23.3 6.4 6.8 4.9 7.3 8.3 5.0 8.2 9.3 5.4 8.8 141-2 3.2 3.2 6.1 4.0 3.76.4 6.8 4.9 7.2 8.0 5.2 8.0 9.4 5.4 8.9 375-2 3.4 3.3 6.4 4.1 3.6 6.46.3 4.5 6.8 8.0 5.0 8.4 9.0 5.0 9.0 ^(a)LM only = L. monocytogenes count^(b)LM + CM = L. monocytogenes count ^(c)CM only = Competitivemicroorganism count

As previously indicated, identification of the nine most antagonisticcultures revealed that six (isolates no. 141-1, 141-2, 143-2, 152, 375-1and 375-2) were Enterococcus durans and 16S rRNA analysis indicated allare indistinguishable; two (isolates no. C-1-92 and C-1-152) wereLactococcus lactis subsp. lactis; and one (isolate no. 143-1) wasLactobacillus plantarum. L. lactis subsp. lactis C-1-92 encoded bothnisA and nisB, but none of the other competitive microorganismsevaluated encoded either nisA or nisB.

The nine antagonistic bacterial isolates and two yeast isolates wereevaluated at about 37° C. at two different cell number combinations(highest level at 6.9 (for yeast) or 8.3-8.4 (for bacteria) log₁₀competitive microorganisms/cm² and 4.6 log₁₀ L. monocytogenes/cm²; andlower level at 6.4-6.5 log₁₀ competitive microorganisms/cm² and 2.9log₁₀ L. monocytogenes/cm²) for their ability to control L.monocytogenes in biofilms on stainless steel coupons. Results of studieswith the highest combination of microbial populations revealed a morethan 6 log₁₀ L. monocytogenes/cm² (to an undetectable level by a directplating method; <1.7 log₁₀ cfu/cm²) differential compared to thepositive control for eight isolates at 37° C. for 24 h and a 3 to 5log₁₀ L. monocytogenes/cm² differential for one isolate, see Table 5below. There was only a 0.2-0.9 log₁₀ L. monocytogenes/cm² differentialfor the two yeast isolates (see Table 5 below). Studies with a lowercombination of microbial populations resulted in all nine competitivebacterial isolates providing a greater than a 6 log₁₀ L.monocytogenes/cm² differential compared to the L. monocytogenes—onlypositive control, see Table 6 below.

TABLE 5 Inhibition at about 37° C. of L. monocytogenes (LM) bycompetitive microorganisms (CM) in biofilms formed on stainless steelcoupons L. monocytogenes or competitive microorganism count (log₁₀cfu/cm²) Trial No. 1 Trial No. 2 0 time 24 h 0 time 24 h Isolate LM LM +CM LM LM + CM LM LM + CM LM LM + CM No. only^(a) CM^(b) only^(c) only CMonly only CM only only CM only Bacteria 141-1 4.6 4.6 8.3 7.8 <1.7^(d)7.5 4.6 4.6 8.3 7.8 <1.7^(d) 7.2 152 4.6 4.6 8.4 7.8 <1.7 7.6 4.6 4.68.3 7.8 <1.7 7.7 C-1-92 4.6 4.6 8.4 7.8 <1.7 6.6 4.6 4.6 8.4 7.8 <1.77.3 143-1 4.6 4.6 8.3 7.8 2.7 7.0 4.6 4.6 8.3 7.8 4.2 7.1 C-1-152 4.64.6 8.4 7.8 <1.7 7.1 4.6 4.6 8.4 7.8 <1.7 7.4 375-1 4.6 4.6 8.3 7.8 <1.77.3 4.6 4.6 8.3 7.8 <1.7 7.7 143-2 4.6 4.6 8.4 7.8 <1.7 6.9 4.6 4.6 8.47.8 <1.7 7.4 141-2 4.6 4.6 8.4 7.8 <1.7 7.4 4.6 4.6 8.4 7.8 <1.7 7.3375-2 4.6 4.6 8.4 7.8 <1.7 7.4 4.6 4.6 8.4 7.8 <1.7 7.5 Yeast C-2-45 4.64.6 6.9 7.8 7.3 5.0 4.6 4.6 6.9 7.8 7.4 7.2 C-3-53 4.6 4.6 6.9 7.8 7.67.3 4.6 4.6 6.9 7.8 6.9 6.3 ^(a)LM only = L. monocytogenes count^(b)LM + CM = L. monocytogenes count ^(c)CM only = Competitivemicroorganism count ^(d)Minimum detection limit 1.7 log₁₀ cfu/cm²

TABLE 6 Inhibition at about 37° C. of L. monocytogenes (LM) bycompetitive microorganism (CM) in biofilms formed on stainless steelcoupons L. monocytogenes or competitive microorganism count (log₁₀cfu/cm²) 0 time 24 h Isolate LM LM + CM LM LM + CM No. only^(a) CM^(b)only^(c) only CM only 141-1 2.9 2.9 6.5 7.7 <1.7^(d) 7.9 152 2.9 2.9 6.67.7 <1.7 7.8 143-1 2.9 2.9 6.4 7.7 <1.7 7.3 C-1-152 2.9 2.9 6.4 7.7 <1.77.8 375-1 2.9 2.9 6.4 7.7 <1.7 8.0 143-2 2.9 2.9 6.5 7.7 <1.7 8.0 141-22.9 2.9 6.5 7.7 <1.7 7.8 375-2 2.9 2.9 6.6 7.7 <1.7 7.9 C-1-92 2.6 2.66.4 7.1 <1.7 7.2 ^(a)LM only = L. monocytogenes count ^(b)LM + CM = L.monocytogenes count ^(c)CM only = Competitive microorganism count^(d)Minimum detection limit 1.7 log₁₀ cfu/cm²

Six competitive bacterial isolates were evaluated under similarconditions (initial cell populations of 3.7 log₁₀ L. monocytogenes/cm²and 6.3-6.5 log₁₀ competitive microorganisms/cm²) at about 15° C., ofwhich two isolates, L. lactis subsp. lactis C-1-92 and C-1-152,controlled L. monocytogenes to an undetectable level (>7.8 log₁₀ L.monocytogenes/cm² differential) through 28 days, which was the end ofthe study, see Table 7 below.

TABLE 7 Inhibition at about 15° C. of L. monocytogenes (LM) bycompetitive microorganisms (CM) in biofilms formed on stainless steelcoupons L. monocytogenes or competitive microorganism count (log₁₀cfu/cm²) 0 time 7 d 14 d 28 d Isolate LM LM + CM LM LM + CM LM LM + CMLM LM + CM No. only^(a) CM^(b) only^(c) only CM only only CM only onlyCM only 141-1 3.7 3.7 6.5 8.5 <1.7^(d) 8.1 9.2 5.3 8.2 9.5 6.6 8.8 1523.7 3.7 6.3 8.5 1.7 7.9 9.2 4.3 8.7 9.5 6.5 8.4 375-1 3.7 3.7 6.4 8.52.7 7.7 9.2 5.3 8.7 9.5 6.6 9.0 C-1-92 3.7 3.7 6.6 8.5 <1.7 6.7 9.2 <1.78.6 9.5 <1.7 8.5 143-1 3.7 3.7 6.5 8.5 4.7 7.7 9.2 2.6 7.8 9.5 6.4 9.0C-1-152 3.7 3.7 6.3 8.5 <1.7 7.5 9.2 <1.7 6.7 9.5 <1.7 8.3 ^(a)LM only =L. monocytogenes count ^(b)LM + CM = L. monocytogenes count ^(c)CM only= Competitive microorganism count ^(d)Minimum detection limit 1.7 log₁₀cfu/cm²

The same six competitive bacterial isolates (at initial populations of6.0-6.7 log₁₀ cfu/cm²) were evaluated in combination with an initialpopulation of 3.7 log₁₀ L. monocytogenes/cm² on stainless steel couponsat about 8° C. Four isolates, E. durans 141-1, 152 and 375-1 and Lc.Lactis subsp. lactis C-1-92, were highly inhibitory to L. monocytogenes,with no listeriae detected (>6.8 log₁₀ cfu/cm² differential) at 21 and28 days, see Table 8 below.

TABLE 8 Inhibition at about 8° C. of L. monocytogenes (LM) bycompetitive microorganisms (CM) in biofilms formed on stainless steelcoupons L. monocytogenes or competitive microorganism count (log₁₀cfu/cm²) 0 time 7 d 14 d 21 d 28 d Isolate LM^(a) LM^(b) + CM^(c) LMLM + CM LM LM + CM LM LM + CM LM LM + CM No. only CM only only CM onlyonly CM only only CM only only CM only 141-1 3.7 3.7 6.0 6.8 3.7 7.4 8.53.3 8.4 8.5 <1.7^(d) 8.0 8.8 <1.7 7.7 152 3.7 3.7 6.4 6.8 3.9 7.5 8.53.5 8.2 8.5 <1.7 7.6 8.8 <1.7 7.8 375-1 3.7 3.7 6.5 6.8 3.7 7.6 8.5 3.08.5 8.5 <1.7 7.8 8.8 <1.7 7.8 C-1-92 3.7 3.7 6.7 6.8 <1.7 4.7 8.5 <1.76.5 8.5 <1.7 5.4 8.8 <1.7 4.7 143-1 3.7 3.7 6.5 6.8 3.9 7.2 8.5 4.4 8.18.5 5.9 7.5 8.8 3.3 7.7 C-1-152 3.7 3.7 6.4 6.8 3.1 7.4 8.5 4.5 8.1 8.54.9 7.8 8.8 6.4 7.7 ^(a)LM only = L. monocytogenes count ^(b)LM + CM =L. monocytogenes count ^(c)CM only = Competitive microorganism count^(d)Minimum detection limit is 1.7 log₁₀ cfu/cm²

Five and six competitive bacterial isolates (at initial populations of6.3-6.6 log₁₀ cfu/cm²) were evaluated in combination with two initialpopulations of L. monocytogenes 2.6 and 4.3 log₁₀ L. monocytogenes/cm²on stainless coupons held at about 4° C. One competitive isolate Lc.Lactis subsp. lactis C-1-92 was especially effective in controlling L.monocytogenes, with no detectable L. monocytogenes (differentialsof >5.1 and >7.0 log₁₀ L. monocytogenes/cm² compared to positive L.monocytogenes—only control) at 35 days when either initial population ofL. monocytogenes was used (see Tables 9 and 10 below). Interestingly,Lc. lactis subsp. lactis C-1-92 did not grow but rather declined in cellnumbers (3.6-3.8 log₁₀ cfu/cm² reduction) during 35 days at about 4° C.,whereas cell populations of all five other competitive microorganismsincreased by 1 to 2 log₁₀ cfu/cm² under the same conditions. The otherfive competitive microorganisms also were inhibitory to L. monocytogenesthrough 35 days at about 4° C., with differentials of L. monocytogenescell populations in biofilms compared to L. monocytogenes—only positivecontrols ranging from 2.0 to >7.0 log₁₀ cfu/cm²

TABLE 9 Inhibition at about 4° C. of L. monocytogenes (LM) bycompetitive microorganisms (CM) in biofilms formed on stainless steelcoupons L. monocytogenes or competitive microorganism count (log₁₀cfu/cm²) 0 time 14 d 21 d 28 d 35 d Isolate LM LM + CM LM LM + CM LMLM + CM LM LM + CM LM LM + CM No. only^(a) CM^(b) only^(c) only CM onlyonly CM only only CM only only CM only 141-1 2.6 2.6 6.4 3.7 2.2 6.4 4.52.3 7.0 6.0 2.3 7.6 6.8 3.2 7.9 152 2.6 2.6 6.6 3.7 <1.7 6.6 4.5 <1.77.3 6.0 2.7 7.8 6.8 2.5 7.8 375-1 2.6 2.6 6.5 3.7 2.2 6.2 4.5 2.2 7.06.0 <1.7 7.3 6.8 2.4 7.8 C-1-92 2.6 2.6 6.3 3.7 <1.7 3.8 4.5 <1.7 3.96.0 <1.7 3.5 6.8 <1.7 2.5 143-1 2.6 2.6 6.5 3.7 <1.7 5.9 4.5 2.2 6.5 6.0<1.7 6.9 6.8 3.0 7.5 C-1-152 2.6 2.6 6.5 3.7 2.0 6.1 4.5 <1.7 6.4 6.02.5 7.2 6.8 3.4 7.4 ^(a)LM only = L. monocytogenes count ^(b)LM + CM =L. monocytogenes count ^(c)CM only = Competitive microorganism count^(d)Minimum detection limit 1.7 log₁₀ cfu/cm²

TABLE 10 Inhibition at about 4° C. of L. monocytogenes (LM) bycompetitive microorganisms (CM) in biofilms formed on stainless steelcoupons L. monocytogenes or competitive microorganism count (log₁₀cfu/cm²) 0 time 14 d 21 d 28 d 35 d Isolate LM LM + CM LM LM + CM LMLM + CM LM LM + CM LM LM + CM No. only^(a) CM^(b) only^(c) only CM onlyonly CM only only CM only only CM only 141-1 4.3 4.3 6.4 3.6 1.7 6.1 5.13.2 6.9 6.6 2.6 7.3 8.7 2.5 8.0 152 4.3 4.3 6.5 3.6 3.0 6.4 5.1 <1.7^(d)6.9 6.6 <1.7 7.6 8.7 <1.7 8.3 C-1-92 4.3 4.3 6.5 3.6 <1.7 4.3 5.1 2.93.9 6.6 2.0 3.5 8.7 <1.7 2.9 143-1 4.3 4.3 6.5 3.6 3.0 6.1 5.1 1.7 6.66.6 4.5 7.0 8.7 4.8 7.4 C-1-152 4.3 4.3 6.6 3.6 <1.7 6.0 5.1 3.1 6.8 6.61.7 7.1 8.7 6.7 7.0 ^(a)LM only = L. monocytogenes count ^(b)LM + CM =L. monocytogenes count ^(c)CM only = Competitive microorganism count^(d)Minimum detection limit 1.7 log₁₀ cfu/cm²

DNA Fingerprinting of the Probiotic Bacteria

As shown in FIG. 1, four probiotic isolates deposited with the ATCC,i.e., C-1-92 [L. lactis subsp. lactis] (lane 2), C-1-152 [L. lactissubsp. lactis] (lane 3), 141-1 [E. durans] (lane 4) and 152 [E. durans](lane 5), were analyzed for their DNA fingerprinting by pulsed field-gelelectrophoresis. Strains were grown in Brain Heart Infusion agar (BHIA)at 37° C. for 16-18 h individually. Bacteria were collected by a cottonswab and suspended in 3 ml of TE (10 mM Tris:1 mM EDTA, pH 8.0).Bacterial concentration was adjusted to an OD reading of 1.0 at 600 nm.240 μl of each bacterial suspension was transferred to a 1.5 ml tube and60 μl of lysozyme solution was added (10 mg/ml). The tubes wereincubated at 37° C. for 10 min and 1.2% SeaKem Gold agarose containingproteinase K (20 mg/ml) was added and mixed with the cell suspension.The melted mixture was transferred to a mold and incubated at roomtemperature for 15 min to solidify. Plugs were then treated for 2 h at54° C. in lysis buffer. Following washing, the plugs were digested with4 μl (40 U/μl) ApaI (Roche Diagnostics Corp., Indianapolis, Ind.) at 30°C. for 16 h and electrophoresed on 1.0% agarose gel in 0.5×Tris-borate-EDTA buffer (0.445 M Tris, 0.0125 M EDTA, and 0.445 M boricacid) with a contour-clamped homogeneous electric field device (CHEFMAPPER, Bio-Rad, Hercule, Calif.). After electrophoresis for 20 h at 6V/cm with pulse times of 4 to 40.01 s at 14° C., the gels were stainedwith ethidium bromide, and the bands were visualized and photographedwith UV transillumination (See FIG. 1).

As discussed above, L. monocytogenes can attach through biofilms tovarious types of surfaces including stainless steel, glass, and rubber.Biofilms, which entrap and protect L. monocytogenes from disinfectants,have been documented in meat and dairy processing plant environments.Furthermore, it is well documented that strains of L. monocytogenes canbecome well established in a food processing environment and remainmembers of the resident microbial flora for many years. As indicatedabove, the present disclosure describes the isolation andcharacterization of a number of microorganisms that (i) thrive incombination with L. monocytogenes within a biofilm at a wide range oftemperatures that occur in food processing facilities (especially underrefrigeration conditions) and (ii) compete to inhibit listeriae growth.In particular, initial screening identified 24 promising candidates withanti-listerial activity. Further competitive testing between thecandidate microorganisms and L. monocytogenes in broth and in biofilmsat different temperatures identified nine bacterial isolates thateffectively reduced, controlled, or eliminated detectable L.monocytogenes depending on environmental conditions. One strain inparticular, Lc. lactis subsp. lactis C-1-92, was especially effective incontrolling L. monocytogenes when in biofilms for extended periods oftime, including at about 4° C. This strain uniquely produced nisin A andnisin B, which are inhibitory to L. monocytogenes. Two other isolates,Enterococcus durans 141-1 and 152, also were very effective incontrolling L. monocytogenes in biofilms. These isolates (E. durans141-1 and 152) grow at refrigeration temperatures and have antagonisticactivity to L. monocytogenes under refrigeration conditions. Thesestrains are useful in food processing locations that require a lowtemperature environment such as for processing ready-to-eat foods.

Application of Probiotic Bacteria in Ready-to-Eat Meat for Reduction ofL. monocytogenes

Three probiotic bacteria, including C-1-92 (Lactococcus lactis subsp.lactis), C-1-152 (Lactococcus lactis subsp. lactis), and 143(Lactobacillus plantarum) were evaluated for their effect to control L.monocytogenes in ready-to-eat meat at a population of 10⁶ cfu/cm². A5-strain mixture of L. monocytogenes (LM113, LM51779, LM112, LM9666 andLM101) including serotype 1/2a, 1/2b and 4b was used as the inoculum.Frankfurters purchased from a local retail store were tested.

Frankfurters were immersed first in a suspension of probiotic bacteria(10⁶ cfu/ml) for 30 seconds and dried in a laminar flow hood for 20minutes, and then immersed in the suspension of L. monocytogenes (10⁴cfu/ml) according to the same procedure.

Two storage temperatures, 4° C. and 8° C., were evaluated. Followinginoculation, each frankfurter was individually sealed in a Ziploc bag,held at 4 and 8° C., and sampled at weekly intervals for the shelf lifeof the product as determined by the use of the date printed on thelabel. Samples were placed in a Whirl-Pak bag containing 10 ml of 0.1%peptone. The bags were agitated on a shaker at 200 rpm for 2 min withintermittent massaging by hands. Sample suspensions were seriallydiluted (1:10) in 0.1% peptone and a volume of 0.1 ml from each dilutiontube was inoculated in duplicate onto modified Oxford agar (MOX) andtryptic soy agar (TSA) plates. The plates were incubated for 24 h at 37°C. Typical colonies (black) on MOX were enumerated as L. monocytogenes.

Results indicated that at 4° C. the treatment by probiotic bacteriareduced the population of L. monocytogenes from 0.7 to 0.8 log₁₀ cfu/cm²(see Table 11 below) and at 8° C. either kept the population at the samelevel or reduced the population of L. monocytogenes from 0.1 to 0.3log₁₀ cfu/cm² (see Table 12 below) depending on different combinationsat the end of the experiment when compared with the population at thebeginning.

TABLE 11 L. monocytogenes counts on frankfurters with and withoutcombinations of probiotic bacteria held at 4° C. Microbial count (log₁₀cfu/ cm²) at week: Treatment 0 1 2 3 4 5 8 L. M. only 3.8 3.9 4.0 4.24.5 4.3 4.2 C-1-92 & 143 + L. M. ^(a) 3.5 3.1 2.7 2.8 2.7 3.2 2.7 C-1-92& C-1-152 + L. M. 3.6 2.8 4.2 3.1 3.1 3.0 2.9 143 & C-1-152 + L. M. 3.63.2 3.0 3.2 3.2 3.0 2.9 C-1-92 & 143 & C-1-152 + L. M. 3.8 3.0 2.7 3.03.1 3.0 3.0 ^(a) L. M. = Listeria monocytogenes.

TABLE 12 L. monocytogenes counts on frankfurters with and withoutcombinations of probiotic bacteria held at 8° C. Microbial count (log₁₀cfu/ cm²) at week: Treatment 0 1 2 3 4 5 L. M ^(a) only 3.8 4.5 4.4 4.74.8 6.9 C-1-92 & 143 + L. M. 3.5 4.2 2.8 4.0 3.5 3.8 C-1-92 & C-1-152 +L. M. 3.6 3.7 3.1 3.6 3.8 3.7 143 & C-1-152 + L. M. 3.6 3.2 2.8 3.1 4.43.7 C-1-92 & 143 & C-1-152 + L. M. 3.8 4.2 2.9 3.2 3.5 3.8 ^(a) L. M. =Listeria monocytogenes.

Application of Probiotic Bacteria in Floor Drains forReduction/Elimination of L. monocytogenes in a Poultry Processing Plant

Two probiotic bacteria, C-1-92 (Lactococcus lactis subsp. lactis) and152 (Enterococcus durans) were selected as the treatment strains and apoultry processing plant was selected for the field trial. Five floordrains in the plant at temperatures ranging from 0.3 to 29° C. wereselected for microbiological observation.

Before the treatment these floor drains were evaluated every two weeksfor five times plus one time after sanitation in the plant for thebaseline determination of L. monocytogenes and aerobic bacterial count.Whirl-Pak “speci-sponge” bags (Nasco, Fort Atkinson, Wis.) were used forcollection of the samples. Sterilized gloves were worn during thecollection and processing of the samples so as to preventcross-contamination. Samples from five locations in and around eachfloor drain were collected. These locations included (i) fluid from thedrain, (ii) the right side of the drain, (iii) the left side of thedrain, (iv) inside of the drain (3.8×7.6 cm), and (v) the surface of thefloor within 1 foot of the drain. The samples were kept at 5° C. andtransported to the laboratory within 12 h and assayed within 72 h. A 10ml volume of brain heart infusion (Becton Dickinson MicrobiologicalSystems, Sparks, Md.) was added into each bag. The bag was individuallypummeled in a stomacher for 1 min at about 130 RPM. The samples werethen serially (1:10) diluted in 0.1% peptone to 10⁻⁸ cfu/ml. A 0.1 mlvolume from each dilution tube was plated on the surface of ModifiedOxford medium (MOX, Oxford Ltd., Basingstoke, Hampshire, UK) and platecount agar (PCA, Becton Dickinson) in duplicate. For counting thebacterial number the MOX plates were incubated at 37° C. for 48 h, andPCA plates were incubated at 30° C. for 48 h. Typical L. monocytogenescolonies (black) were counted as presumptive L. monocytogenes. Up tofive colonies from the highest dilution were randomly picked forconfirmation of L. monocytogenes by a latex precipitation assay (Oxoid).

A population of 10⁷ probiotic bacteria/ml in a one-time application offoam formula were used to treat the floor drain after the sanitationprocess was finished daily for four times in a first week (Mondaythrough Thursday). Then the treatment was performed twice a week(Tuesday and Thursday) for the next three weeks. Samples were collectedonce a week for the five weeks after the treatment was started. Bacteriawere individually grown in 300 ml Lactobacilli MRS broth (MRS, BectonDickinson) at 32° C. for 24 h. The bacterial broth was precipitated at10,000×g for 20 min at 4° C. The bacteria were then resuspended in 25 mlMRS broth at about 10⁹ cfu/ml. A volume of 1 ml was serially (1:10)diluted in 0.1% peptone to 10⁻⁸ cfu/ml. A quantity of 0.1 ml fromdilution tubes (10⁻⁵ to 10⁻⁸) was plated on MRS agar and tryptic soyagar in duplicate for the actual counting of the bacterial number. Afterarriving at the processing plant, the two isolates (25 ml each), 20 mlof Dy-gest I, 20 ml of Dy-gest II, plus 1 gallon of water were added tothe tank foamer (Ecolab, St. Paul, Minn.). After connecting the airsupply to the tank foamer, the foam was applied in each floor drain.

Results demonstrated that the average number of L. monocytogenes infloor drains sampled at six different times (at two week intervals) inthis poultry processing plant before the treatment by probiotic bacteriaranged from 3.3 to 4.0 log₁₀ cfu/cm² for drain #1, from 4.2 to 5.4 log₁₀cfu/cm² for drain #3, from 3.4 to 4.5 log₁₀ cfu/cm² for drain #4, from3.2 to 4.2 log₁₀ cfu/cm² for drain #6, and from 6.1 to 8.2 log₁₀ cfu/cm²for drain #8 (see Table 13 below).

TABLE 13 The mean and standard deviation of L. monocytogenes count(log₁₀ cfu/cm²) from drain samples located at different temperatures andcollected at six different times Floor drains Location #1 #3 #4 #6 #8Drain 3.3 ± 0.8 4.2 ± 1.2 4.2 ± 1.1 4.2 ± 1.2 7.6 ± 1.1 Right side 3.9 ±0.7 5.0 ± 0.9 4.3 ± 0.8 3.5 ± 1.9 7.6 ± 0.8 Left side 4.0 ± 1.0 4.4 ±1.3 3.4 ± 1.1 3.2 ± 1.5 8.2 ± 0.5 Inside 3.5 ± 0.8 5.4 ± 1.3 4.5 ± 1.13.6 ± 1.3 7.8 ± 0.9 Floor (1 foot) 3.6 ± 0.9 5.3 ± 0.6 4.3 ± 1.4 3.3 ±1.3 6.1 ± 1.9 Temperature 21.8 ± 2.9  18.6 ± 2.3  4.7 ± 1.3   5 ± 1.228.6 ± 0.9 After the treatment by these two probiotic bacteria, the average numberof L. monocytogenes ranged from 1.8 to 2.0 log₁₀ cfu/cm² for drain #1,1.9 to 2.8 log₁₀ Cfu/cm² for drain #3, 1.7 to 2.1 log₁₀ cfu/cm² fordrain #4, 1.9 to 2.1 log₁₀ cfu/cm² for drain #6 and 3.5 to 4.0 log₁₀cfu/cm² for drain #8 (see Table 14 below).

TABLE 14 The mean and standard deviation of L. monocytogenes count(log₁₀ cfu/cm²) from drain samples collected after probiotic treatmentat five different times Floor drains Location #1 #3 #4 #6 #8 Drain 1.8 ±0.4 1.9 ± 0.3 1.9 ± 0.3 2.1 ± 0.4 4.0 ± 2.0 Right side 2.0 ± 0.5 2.1 ±0.8 1.7 ± 0.1 1.9 ± 0.7 3.9 ± 2.0 Left side 2.0 ± 0.6 2.3 ± 0.6 2.1 ±1.2 1.9 ± 0.4 3.9 ± 2.2 Inside 2.0 ± 0.5 2.8 ± 1.4 1.7 ± 0.1 2.0 ± 0.73.7 ± 1.8 Floor (1 foot) 1.9 ± 0.5 2.1 ± 0.6 2.1 ± 0.9 1.9 ± 0.6 3.5 ±0.8 Temperature 11.7 ± 2.2  11.5 ± 2.2  0.9 ± 1.0 2.5 ± 0.9 23.5 ± 1.5 

Compared with the L. monocytogenes counts before the treatment, theaverage population of L. monocytogenes after the probiotic treatment wasreduced 1.7 log₁₀ cfu/cm² for drain #1, 2.6 log₁₀ cfu/cm² for drain #3,2.2 log₁₀ cfu/cm² for drain #4, 1.6 log₁₀ cfu/cm² for drain #6 and 3.7log₁₀ cfu/cm² for drain #8. These results demonstrated that theapplication of these two probiotic bacteria significantly reduced thepopulation of L. monocytogenes in the floor drains located at varioustemperatures in this poultry processing plant.

Accordingly, in light of the above discussion, it should be appreciatedthat compositions for, and methods of, treating one or more surfaces ofa food processing facility are provided. For example, one or moresurfaces in a facility that processes substances consumed for theirnutritive and/or recreational value (e.g., alcoholic beverages) can betreated by the methods described herein. It should be understood thatany surface of a food processing facility that serves, or potentiallycould serve, as a point of contamination for the processing plantenvironment and/or food products can be treated by the methods describedherein. For example, these surfaces include, but are not limited to,surfaces included in the plumbing system of a food processing facility(e.g., drain surfaces), surfaces of food processing equipment, andstructural surfaces of a food processing facility.

In addition, it should be appreciated that compositions for, and methodsof, treating a food product (e.g., a food product can be a substanceconsumed or eaten for its nutritive and/or recreational value) areprovided. For example, a method of treating a food product of thepresent invention can include placing the food product in contact withone or more probiotic bacteria (e.g., competitive exclusionmicroorganisms) described herein. It should be understood that anyportion of a food product that serves, or potentially could serve, as apoint of contamination for the food product can be treated by themethods described herein.

The methods described herein inhibit the growth of undesirablemicroorganisms on such surfaces of food processing facilities and onfood products. For example, inhibiting the growth of undesirablemicroorganisms (e.g., L. monocytogenes) includes, but is not limited to,the killing of, decreasing or stopping the growth of, exclusion of, orany other mechanism by which growth of undesirable microorganisms iscontrolled.

One illustrative method contemplated for treating a surface of a foodprocessing facility or food product which has, or could have, a firstpopulation of microorganisms disposed thereon includes inoculating thesurface with a composition that includes a second population ofmicroorganisms. In one embodiment, the second population ofmicroorganisms can be disposed in a biofilm. For example, anillustrative method includes disposing a biofilm containing a secondpopulation of microorganisms onto a surface, where the second populationof microorganisms is inhibitory or bactericidal to the first populationso that when the second population is placed in the presence of thefirst population the second populations inhibits the growth of the firstpopulation of microorganisms. Accordingly, it should be appreciated thata goal of the aforementioned inoculation is to contact a surface with asufficient quantity of biofilm so that a second population ofmicroorganisms contained in the biofilm can colonize the surface andinhibit the growth of a first population of microorganisms. It is alsocontemplated that, initially, the second population of microorganisms isnot contained in a biofilm, but is capable of forming a biofilm afterbeing placed on the surface. Therefore, this type of microorganism(i.e., the second population of microorganism) would initially not becontained in a biofilm when placed on the surface, but would thereafterform one to be contained in. It should be appreciated that having aprobiotic microorganism, such as a competitive microorganism, in abiofilm facilitates the competitive microorganism's ability to inhibitthe growth of undesirable microorganisms since the biofilm enables thecompetitive microorganism to adhere to surfaces for an extended periodof time and not be washed away or significantly damaged by routinesanitation procedures. However, it should be appreciated that a biofilmis not absolutely required, and a goal of the aforementioned inoculationcan be to contact a surface with a sufficient quantity of the secondmicroorganism so that the second microorganism can colonize the surfaceand inhibit the growth of the first of microorganism in the absence of abiofilm. The above described methods and techniques discussed inreference to a surface in a food processing facility are also applicableto inhibiting the growth of undesirable microorganisms on the surfacesof food products.

As indicated above, a non-limiting example of a first population ofmicroorganisms is one that includes L. monocytogenes, while examples ofsecond populations of microorganisms include, but are not limited to,those competitive microorganisms discussed above which possessantagonistic activity to L. monocytogenes. In particular, with respectto controlling the growth of L. monocytogenes, the second population ofmicroorganisms can include Enterococcus durans 141-1 having ATCCaccession number PTA-4758, Enterococcus durans 152 having ATCC accessionnumber PTA-4759, Lactococcus lactis C-1-92 having ATCC accession numberPTA-4760, or Lactococcus lactis C-1-152 having ATCC accession numberPTA-4761.

In addition to the subject matter discussed above, a kit for inhibitingthe growth of or killing a first microorganism population disposed on asurface is contemplated. An illustrative example of such a kit caninclude a container containing a biofilm with a second microorganismpopulation disposed in the biofilm. The second microorganism populationis inhibitory to the first microorganism population so that when thesurface is inoculated with the biofilm (i.e., the second microorganismpopulation is placed in the presence of said first microorganismpopulation) the second microorganism population inhibits the growth ofthe first population of microorganisms. In an alternative embodiment,the kit can include one container with a biofilm disposed therein andanother container with the second microorganism population disposedtherein. The second microorganism population can be mixed into thebiofilm just prior to inoculating the surface, or the biofilm can bedisposed onto the surface first, followed by the disposing of the secondmicroorganism population into the biofilm. In another alternative, thekit can include, as discussed above, a second microorganism populationcapable of forming a biofilm after being disposed on the surface.Accordingly, this type of second microorganism population initially isnot contained in a biofilm when place on the surface, but thereafterforms one to be contained in. As indicated above, a non-limiting exampleof a first population of microorganisms is one that includes L.monocytogenes, while examples of second populations of microorganismswhich can be utilized in the kit include, but are not limited to, thosecompetitive microorganisms discussed above which possess antagonisticactivity to L. monocytogenes.

It should also be appreciated that the present disclosure also providesa method of selecting a population of inhibitory bacteria. Anillustrative example of one such method includes (a) isolatingnaturally-occurring bacteria populations from a food processingfacility, (b) culturing the isolated naturally-occurring bacteriapopulations; and (c) testing each isolated naturally-occurring bacteriapopulation for the ability to inhibit the growth of a microorganism,where the isolated naturally-occurring bacteria populations having theability to inhibit the growth of the microorganism are identified as apopulation of inhibitory bacteria. As indicated herein, one exemplarymicroorganism the aforementioned selecting method can be utilized for isL. monocytogenes.

While the invention has been illustrated and described in detail in theforegoing description, such an illustration and description is to beconsidered exemplary and not restrictive in character, it beingunderstood that only the illustrative embodiments have been shown anddescribed and that all changes and modifications within the spirit ofthe invention are desired to be protected.

There are a plurality of advantages of the present invention arisingfrom the various features of the invention described herein. It will benoted that alternative embodiments of the present invention may notinclude all of the features described, but yet still benefit from atleast some of the advantages of such features.

1. A composition comprising a biologically pure culture of bacteriacomprising a species selected from the group consisting followingEnterococcus durans having ATCC accession number PTA-4758, Enterococcusdurans having ATCC accession number PTA-4759, Lactococcus lactis havingATCC accession number PTA-4760, and Lactococcus lactis having ATCCaccession number PTA-4761.
 2. The composition of claim 1 wherein saidcomposition comprises two biologically pure cultures of bacteriaselected from the group consisting following Enterococcus durans havingATCC accession number PTA-4758, Enterococcus durans having ATCCaccession number PTA-4759, Lactococcus lactis having ATCC accessionnumber PTA-4760, and Lactococcus lactis having ATCC accession numberPTA-4761.
 3. The composition of claim 2 wherein said compositioncomprises biologically pure cultures of Enterococcus durans having ATCCaccession number PTA-4759, and Lactococcus lactis having ATCC accessionnumber PTA-4760.
 4. The composition of claim 1 wherein said biologicallypure culture of bacteria is disposed in a biofilm.
 5. A kit forinhibiting the growth of a first microorganism population disposed on asurface, said kit comprising: a biofilm; and a second microorganismpopulation comprising a biologically pure culture of bacteria comprisinga species selected from the group consisting following Enterococcusdurans having ATCC accession number PTA-4758, Enterococcus durans havingATCC accession number PTA-4759, Lactococcus lactis having ATCC accessionnumber PTA-4760, and Lactococcus lactis having ATCC accession numberPTA-4761.
 6. The kit of claim 5 wherein said second microorganismpopulation is disposed in said biofilm.
 7. The kit of claim 5 whereinsaid second microorganism population comprises a mixture of twobiologically pure cultures of bacteria selected from the groupconsisting following Enterococcus durans having ATCC accession numberPTA-4758, Enterococcus durans having ATCC accession number PTA-4759,Lactococcus lactis having ATCC accession number PTA-4760, andLactococcus lactis having ATCC accession number PTA-4761.
 8. Aninoculant composition, comprising: a biofilm having disposed therein apurified bacteria selected from the group consisting of Enterococcusdurans having ATCC accession number PTA-4758, Enterococcus durans havingATCC accession number PTA-4759, Lactococcus lactis having ATCC accessionnumber PTA-4760, and Lactococcus lactis having ATCC accession numberPTA-4761.
 9. A method for selecting bacteria which inhibit the growth ofListeria monocytogenes, comprising: (a) isolating naturally-occurringbacteria from a food processing facility; (b) culturing said isolatednaturally-occurring bacteria; and (c) testing said isolatednaturally-occurring bacteria for the ability to inhibit the growth ofListeria monocytogenes.
 10. The method of claim 9, wherein: saidnaturally-occurring bacteria are isolated from a drain of said foodprocessing facility.
 11. The method of claim 9 comprising the additionalsteps of depositing said naturally-occurring bacteria and said Listeriamonocytogenes in a biofilm; placing the biofilm on a stainless steelcoupon; co-culturing the bacteria for a predetermined length of time at4° C., 8° C., 15° C. and 37° C.; removing weakly adherent bacteria wereby washing; determining total bacterial counts of the bacteria remainingafter said washing step.